A 25-kDa antioxidant enzyme from S. cerevisiae, which furnishes the protection against oxidation system capable of generating reactive oxygen species, has recently been identified as a thioredoxin-dependent peroxidase (TPx). At least five different TPx homologs exist in mammalian tissues. But none of them have been associated with known biochemical functions. Four out of the five mammalian homologs, NKEF-A, NKEF-B, MER5, and Orf-06 were expressed in E. coli and purified. All the recombinant protein provided the protection against thiol-containing metal catalyzed oxidation system, but not against the oxidation system containing ascorbate. Recombinant NKEF-A, NKEF-B, MER5 exhibited peroxidase activity when the reducing equivalents were provided with thioredoxin system containing thioredoxin, thioredoxin reductase and NADPH but not when the reducing equivalents were provided with glutaredoxin system containing glutaredoxin, glutathione, glutathione reductase, and NADPH. Orf-O6, like NKEF-A, NKEF-B, and MER5, reduces peroxides in the presence of nonphysiological hydrogen donor dithiothreitol but not in the presence of the thioredoxin system or glutaredoxin system. Thus ,the physiological hydrogen donor for Orf-06 remains to be identified. Kinetic parameters of NKEF-A, NKEF-B, MER5 were evaluated to assess their catalytic efficiency. MER5 represented the highest peroxidase activity (11 mu mole/min/mg) followed by NKEF-A (5.6 mu mole/min/mg) and NKEF-B (3 mu mole/min/mg). The apparent Km vales for H2O2 were < 10 muM for all three enzymes. The catalytic efficiencies (kcat/Km) are >107 M-1s-1. Furthermore, immunoblot analysis suggests that these TPx homologs are ubiquitous an abundant proteins in mammalian tissues; e.g., the amount of each isotype ranges from 0.05 to 0.4 % of total cytosolic protein in K-562 human myelogenous leukemia cells. Thus, total catalytic efficiency (kcat/Kmx [E]) contributed by these TPx homologs may exceed those contributed by catalase or glutathione peroxidase in the cytosol of most mammalian cells.